Glucose transport occurs in the adipose cell through a highly specific facilitated diffusion process. Insulin, in turn, increases the maximum transport velocity. Characterization of the adipose cell's transporter proteins has been initiated by developing an equilibrium 3H-cytochalasin B binding assay for quantitating the number of functional glucose transport systems in the purified plasma membranes of isolated rat cells which have been preincubated in the absence or presence of insulin. Cytochalasin B rapidly and reversibly binds to adipose cell plasma membranes in a saturable fashion. A small, but highly specific, fraction of this binding is competitively inhibited by D-, but not only L-, glucose. Curve fitting analysis of cytochalasin B binding isotherms in the absence and presence of D-glucose permits identification of a single class of D-glucose inhibitable binding sites with a dissociation constant of 120 nM, a value comparable to the inhibitory constant of cytochalasin B for glucose transport in these membranes. These sites appear, therefore, to represent the glucose transporter. Insulin increases cytochalasin B binding by increasing the number of D-glucose inhibitable sites, and presumably, functional glucose transport systems, in rough proportion to insulin's stimulatory effect on transport activity in the intact cell.